1. Field of the Invention
The present invention relates to chimeric lyssavirus nucleic acids, and chimeric polypeptides and proteins encoded by these nucleic acids. More particularly, the invention relates to chimeric lyssavirus nucleic acids and proteins that can be used in immunogenic compositions, such as vaccines. Thus, the invention also relates to carrier molecules for expressing chimeric lyssavirus nucleic acids, methods of producing chimeric lyssavirus proteins and polypeptides, and methods of treating individuals to ameliorate, cure, or protect against lyssavirus infection. The compositions of the invention can also be used to express peptides, polypeptides, or proteins from organisms other than lyssaviruses. Thus, the invention provides methods of treating individuals to ameliorate, cure, or protect against many different infections, diseases, and disorders.
2. Background of the Related Art
Rabies is an encephalopathic disease caused by members of the Lyssavirus genus Within the Rhabdoviridae family. Rabies infects all warm-blooded animals and is almost invariably fatal in humans if not treated. On the basis of nucleotide sequence comparisons and phylogenetic analyses, the Lyssavirus genus has been divided into 7 genotypes (GT). GT1 includes the classical rabies viruses and vaccine strains, whereas GT2 to GT7 correspond to rabies-related viruses including Lagos bat virus (GT2); Mokola virus (GT3); Duvenhage virus (GT4); European bat lyssavirus 1 (EBL-1: GT5); European bat lyssavirus 2 (EBL-2: GT6); and Australian bat lyssavirus (GT7).
Based on antigenicity, the Lyssavirus genus was first divided into four serotypes. More recently, this genus was divided into two principal groups according to the cross-reactivity of virus neutralizing antibody (VNAb): Group 1 consists of GT1, GT4, GT5, GT6, and GT7, while Group 2 consists of GT2 and GT3. Viruses of group 2 are not pathogenic when injected it peripherally in mice. Virulence of lyssaviruses is dependent, at least in part, on the glycoprotein present in the viral coat. Interestingly, the glycoproteins of group 2 viruses show a high degree of identity, in the region containing amino acids that play a key role in pathogenicity, to the corresponding sequence of avirulent GT1 viruses (see, for example, Coulon et al., 1998, “An avirulent mutant of rabies virus is unable to infect motoneurons in vivo and in vitro”, J. Virol. 72:273–278).
Rabies virus glycoprotein (G) is composed of a cytoplasmic domain, a transmembrane domain, and an ectodomain. The glycoprotein is a trimer, with the ectodomains exposed at the virus surface. The ectodomain is involved in the induction of both VNAb production and protection after vaccination, both pre- and post-exposure to the virus. Therefore, much attention has been focused on G in the development of rabies subunit vaccines. Structurally, G contains three regions, the amino-terminal (N-terminal) region, a “hinge” or “linker” region, and the carboxy-terminal (C-terminal) region. (See FIG. 1.)
As depicted in FIG. 1, it is generally thought that the glycoprotein (G) ectodomain has two major antigenic sites, site II and site III, which are recognized by about 72.5% (site II) and 24% (site III) of neutralizing monoclonal antibodies (MAb), respectively. The site II is located in the N-terminal half of the protein and the site III is located in the C-terminal half of the protein. The two halves are separated by a flexible hinge around the linear region (amino acid 253 to 257).
The G ectodomain further contains one minor site (site a), and several epitopes recognized by single MAbs (I: amino acid residue 231 is part of the epitope; V: residue 294 is part of the epitope, and VI: residue 264 is part of the epitope) (Benmansour et al., 1991, “Antigenicity of rabies virus glycoprotein”, J. Virol. 65:4198–4203; Dietzschold et al., 1990, “Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine”, J. Virol. 64:3804–3809; Lafay et al., 1996, “Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites”, J. Gen. Virol. 77:339–346; Lafon et al., 1983, “Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies”, J. Gen. Virol. 64:843–845). Site II is conformational and discontinuous (amino acid residues 34 to 42 and amino acid residues 198 to 200, which are associated by disulfide bridges), whereas site III is conformational and continuous (residues 330 to 338). Lysine 330 and arginine 333 in site III play a key role in neurovirulence and may be involved in the recognition of neuronal receptors (see, for example, Coulon et al., supra, and Tuffereau et al., 1998, “Neuronal cell surface molecules mediate specific binding to rabies virus glycoprotein expressed by a recombinant baculovirus on the surfaces of lepidopteran cells”, J. Virol. 72:1085–1091). Sites II and III seem to be close to one another in the three dimensional structure and exposed at the surface of the protein (Gaudin, Y., 1997, “Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones”, J. Virol. 71:3742–3750). However, at low pH, the G molecule takes on a fusion-inactive conformation in which site II is not accessible to MAbs, whereas sites a and III remain more or less exposed (Gaudin, Y. et al., 1995, “Biological function of the low-pH, fusion-inactive conformation of rabies virus glycoprotein (G): G is transported in a fusion-inactive state-like conformation”, J. Virol. 69:5528–5533; Gaudin, Y., et al., 1991, “Reversible conformational changes and fusion activity of rabies virus glycoprotein”, J. Virol. 65:4853–4859).
Moreover, several regions distributed along the ectodomain are involved in the induction of T helper (Th) cells (MacFarlan, R. et al., 1984, “T cell responses to cleaved rabies virus glycoprotein and to synthetic peptides”, J. Immunol. 133:2748–2752; Wunner, W. et al, 1985, “Localization of immunogenic domains on the rabies virus glycoprotein”, Ann. Inst. Pasteur, 136 E:353–362). Based on these structural and immunological properties, it has been suggested that the G molecule may contain two immunologically active parts, each potentially able to induce both VNAb and Th cells (Bahloul, C. et al, 1998, “DNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssaviruses”, Vaccine 16:417–425).
Currently available vaccines predominantly consist of, or are derived from, GT1 viruses, against which they give protection. Many vaccine strains are not effective against GT4, and none are effective against GT2 or GT3. However, the protection elicited against GT4 through 6 depends on the vaccine strain. For example, protection from the European bat lyssaviruses (GT5 and GT6), the isolation of which has become more frequent in recent years, by rabies vaccine strain PM (Pitman-Moore) is not robust. Strain PM induces a weaker protection against EBL1 (GT5) than the protection it provides against strain PV (Pasteur virus).
Because, in part, of the importance of rabies in world health, there is a continuing need to provide safe, effective, fast-acting vaccines and immunogenic compositions to treat and prevent this disease. Many approaches other than use of whole-virus preparations have been proposed and/or pursued to provide an effective, cost-efficient immunogenic composition specific for rabies viruses. For example, as discussed above, subunit vaccines have been developed. Also, vaccines that could generate an immune response to multiple rabies serotypes as well as various other pathogens has been proposed as having some value (European Commission COST/STD-3, 1996, “Advantages of combined vaccines”, Vaccine 14:693–700). In fact, use of a combined vaccine of diphtheria, tetanus, whole cell pertussis, inactivated poliomyelitis, and rabies has recently been reported (Lang, J. et al., 1997, “Randomised Feasibility trial of pre-exposure rabies vaccination with DTP-IPV in infants”, The Lancet 349:1663–1665). Combined vaccines including rabies have also been used for immunization of dogs (distemper, hepatitis, leptospirosis, and parvo-canine viruses), cats (panleukopenia, calici- and parvo-feline viruses), and cattle (foot and mouth disease virus) (Pastoret, P-P. et al., 1997, “Vaccination against rabies”, In Veterinary Vaccinology, Pastoret, P-P. et al., Eds. (Elsevier): 616–628).
Moreover, vaccines produced in tissue culture are expensive to produce despite some attempts to reduce their cost. Consequently DNA vaccines, which are less expensive to produce and offer many advantages, would constitute a valuable alternative. Reports of DNA vaccinations include mouse inoculation with plasmids containing the gene encoding the rabies virus glycoprotein (G). Such inoculation is very potent in inducing humoral and cellular immune responses in association with protection against an intracerebral challenge (see, for example, Lodmell, D. et al., 1998, “DNA immunization protects nonhuman primates against rabies virus”, Science Med. 4:949–952; Xiang, Z. et al., 1994, “Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus”, Virol. 199:132–140; and Lodmell, D. et al., 1998, “Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection”, Vaccine 16, 115). DNA immunization can also protect nonhuman primates against rabies (Lodmell et al, 1998, supra).
Because administration of plasmid DNA generates humoral and cellular immune responses, including cytotoxic T-Lymphocyte (CTL) production (for review see Donnelly, J. et al., 1997, “DNA Vaccines”, Annu. Rev. Immunol. 15:617–648) and is based on a versatile technology, immunization with plasmid DNA may offer a satisfying prospect for multivalent vaccines. However, the use of a mixture of plasmids or a single plasmid expressing several antigens is believed to induce interference problems at both transcriptional and immunological levels (Thomson, S. et al., 1998, “Delivery of multiple CD8 cytotoxic cell epitopes by DNA vaccination”, J. Immunol. 160: 1717–1723). Therefore, there exists a need to develop and produce multivalent DNA-based vaccines that are effective against rabies and various other diseases; that are safe; and that are cost-efficient to produce and use.